A platform for genome mining of multidrug-resistant pathogens to develop therapeutic phages using synthetic biology

利用合成生物学开发治疗性噬菌体的多重耐药病原体基因组挖掘平台

• 批准号: 10356122
• 负责人: Yoann Stephane Le Breton
• 金额: $ 16.91万
• 依托单位: GENEVA FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-18 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10356122
• 关键词: Address; Antibiotic Resistance; Antibiotics; Antimicrobial Resistance; Bacteria; Bacteriophages; Biology; Biomedical Engineering; Cell Surface Receptors; Cells; Centers for Disease Control and Prevention (U.S.); Chromosomes; Cytolysis; DNA sequencing; Development; Disease; Endotoxins; Engineering; Environment; Essential Genes; Experimental Designs; Genes; Genetic; Genetic Engineering; Genome; Genome engineering; Goals; Gold; Horizontal Gene Transfer; Human; Infection; Knowledge; Learning; Life; Life Cycle Stages; Lytic; Lytic Phase; Medical; Methods; Microbial Biofilms; Mining; Modality; Modeling; Modern Medicine; Modification; Monoclonal Antibodies; Multi-Drug Resistance; Mutation; Patients; Penicillin Resistance; Pharmaceutical Preparations; Preparation; Production; Property; Prophages; Reporting; Resistance; Rogaine; Safety; Sepsis; Series; Source; Specificity; Streptococcal Infections; Streptococcus pyogenes; Structure; Testing; Therapeutic; Vaccines; Virulence; Virulence Factors; Wound Infection; antimicrobial; arm; bacterial resistance; base; design; design-build-test; efficacy testing; experience; functional genomics; gene interaction; global health; high throughput analysis; human pathogen; in vivo; in vivo Model; insight; interest; multi-drug resistant pathogen; pathogen; pathogenic bacteria; receptor; research and development; small molecule; synthetic biology; therapeutic target; tool

PROJECT SUMMARY/ABSTRACT Multidrug resistant [MDR] pathogens represent a global health threat and a challenge for modern medicine; and, as bacterial resistance to new antibiotics is now outpacing the antibiotic development effort, it is critical to develop new effective antimicrobial alternatives. The antimicrobial resistance crisis is bringing new interests worldwide to develop phage-based therapies. Over the last decade, efforts in the U.S. to produce phage therapeutics targeting different bacterial pathogens have shown promising results, including successful treatments of life-threatening infections in human patients. Safety of phage therapy is still a concern in the U.S.; however FDA has highlighted requirements for phage preparation: they need to be safe, pure, potent, exclusively lytic, non-transducing, and lacking undesirable genes (antibiotic resistance, virulence factors) and bacterial endotoxins. If bacteriophages for therapy have historically been isolated from natural environments, recent progresses in phage genetics and genome engineering have proven successful to generate synthetic, strictly lytic derivatives targeting pathogens. The development of synthetic phages against MDR pathogens would require pipelines to accelerate our knowledge on newly discovered phages and their potential for synthetic biology. Critical insights into their biology, i.e. genome structure, phage replication cycle, genetic content (essential genes versus dispensable [antibiotic resistance and virulence genes]), interaction with the target host, are a prerequisite. Here, we propose a platform to (i) mine the genomes of MDR pathogens, a gold mine to identify dormant lysogenic phages directly from within their natural host; and (ii) develop high-throughput pipelines to quickly gain knowledge on the phage biology to guide our efforts to engineer synthetic phages as therapeutics. We will use the Group A Streptococcus (GAS), a “Concerning Threat” on the 2019 CDC “18 MDR pathogens” Watch List, as our model. We showed that Tn-seq could identify functional lysogenic phages from cryptic ones in GAS genomes, and mutations to reboot dormant prophages into their lytic cycle. In Aim 1, we will produce the critical knowledge to guide decision on what phages to select for therapeutic potential using synthetic biology: we will experimentally assess phage genome organization, phage replication/transduction mechanisms, host range and cell surface receptor(s). In Aim 2, we will implement a design-build-test-learn cycle" pipeline to optimize the synthetic biology effort, i.e. deletion of undesirable genes and addition of “payload” genes, to enhance their potential as therapy phages. Finally, we will use in vivo model of wound infection to test the efficacy of the synthetic phages we generated. Our overarching goal is to develop the tools and experience to apply our synthetic biology phage-engineering platform to other MDR pathogens.

项目总结/摘要 多药耐药病原体是一种全球性的健康威胁，也是对现代医学的挑战; 而且，由于细菌对新抗生素的耐药性现在超过了抗生素开发的努力， 开发新的有效的抗菌替代品。抗生素耐药性危机带来了新的利益 开发基于噬菌体的疗法。在过去的十年里，美国生产噬菌体的努力 靶向不同细菌病原体的治疗剂已经显示出有希望的结果，包括成功的 治疗人类患者的危及生命的感染。噬菌体治疗的安全性仍然是一个问题， 美国;然而，FDA强调了对噬菌体制备的要求：它们需要是安全、纯、有效的， 仅裂解、非转导和缺乏不良基因（抗生素抗性、毒力因子）， 细菌内毒素 如果用于治疗的噬菌体在历史上是从自然环境中分离出来的，那么最近的进展是， 噬菌体遗传学和基因组工程已被证明成功地产生了合成的、严格裂解的衍生物 针对病原体。针对MDR病原体的合成抗生素的开发将需要管道， 加速我们对新发现的生物学及其合成生物学潜力的了解。重要见解 它们的生物学，即基因组结构，噬菌体复制周期，遗传内容（必需基因与 抗生素抗性和毒力基因）与靶宿主的相互作用是先决条件。 在这里，我们提出了一个平台，（i）挖掘MDR病原体的基因组，这是一个金矿，可以识别休眠的 直接从其天然宿主内溶原性大肠杆菌;和（ii）开发高通量管道， 获得有关噬菌体生物学的知识，以指导我们努力设计合成的噬菌体疗法。 我们将使用A组链球菌（GAS），这是2019年CDC“18种MDR病原体”上的“关注威胁” 观察名单，作为我们的模型。我们发现Tn-seq可以从隐蔽的细胞中识别出功能性溶原性细胞 GAS基因组中的突变，以及使休眠的前噬菌体重新进入其裂解周期的突变。在目标1中，我们将 关键知识，以指导决定选择何种药物的治疗潜力，使用合成 生物学：我们将通过实验评估噬菌体基因组组织、噬菌体复制/转导 机制、宿主范围和细胞表面受体。在目标2中，我们将实现设计-构建-测试-学习 优化合成生物学工作的“循环”管道，即删除不需要的基因和添加 “有效载荷”基因，以增强其作为治疗药物的潜力。最后，我们将使用在体创伤模型 来测试我们合成的病毒的效力。我们的首要目标是开发工具 以及将我们的合成生物学噬菌体工程平台应用于其他MDR病原体的经验。